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This document outlines an experiment to study bacterial morphology through simple staining and Gram staining. It describes the basic procedures for both simple staining and Gram staining of bacterial cultures. Simple staining involves using a single dye like methylene blue to color bacteria based on their slight negative charge. Gram staining distinguishes between Gram positive and Gram negative bacteria based on differences in their cell wall structure that cause them to retain or release the crystal violet/iodine complex. The experiment aims to familiarize students with basic staining techniques used in microbiology to identify unknown bacterial cultures.

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Aditya Murti
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30 views

bbl132 3

This document outlines an experiment to study bacterial morphology through simple staining and Gram staining. It describes the basic procedures for both simple staining and Gram staining of bacterial cultures. Simple staining involves using a single dye like methylene blue to color bacteria based on their slight negative charge. Gram staining distinguishes between Gram positive and Gram negative bacteria based on differences in their cell wall structure that cause them to retain or release the crystal violet/iodine complex. The experiment aims to familiarize students with basic staining techniques used in microbiology to identify unknown bacterial cultures.

Uploaded by

Aditya Murti
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
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Indian Institute of Technology Delhi

Department of Biochemical Engineering and Biotechnology

I SEMESTER
BBL132 – GENERAL MICROBIOLOGY
LABORATORY

EXPERIMENT # 3
________________________________________________________________________________

AIM

To study morphology of bacterial organisms by simple staining and to perform Gram staining of the
given bacterial cultures.

BACKGROUND

Simple staining of bacterial organisms is very important starting point for identifying species. In this
procedure, single stain is used to colour a bacterial organism. Most commonly used dye for simple
staining is methylene blue, crystal violet, and basic fuchsin. These dyes contain colour-bearing ions
(chromophores) and are positively charged (cationic). As bacteria are slightly negatively charged,
they produce attraction with cationic chromophores of dyes called basic dyes.

Gram staining method is named after a Danish bacteriologist Hans Christian Gram (1882). It is one
of the most important staining techniques in Microbiology and is usually the first step in
identification of unknown culture. It is also used for the classification of bacteria into two general
groups, namely, Gram positive and Gram negative.

MATERIALS REQUIRED

Microbial cultures, stain – methylene blue, basic fushein and crystal violet, ethanol, iodine, safarnin,
distilled water, tissue paper, oil immersion, grease-free slides, microscope, etc

PROCEDURE FOR SIMPLE STAINING

1) Prepare a smear of culture on the microscopic slide and air dry.


2) Heat-fix cells by passing the slide rapidly across the flame (do not overheat as it may shrink the
cells and distort the shape)
3) Flood the slide with methylene blue and allow it to react for 1 minute.
4) Tilt the slide and wash with distilled water to remove excess stain (be gentle).
5) Blot off excess water and allow it to dry completely.
6) Carefully position the oil immersion objective over the smear after adding immersion oil to the
smear.

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PROCEDURE FOR GRAM STAINING

1) Prepare a smear of culture on the microscopic slide and air dry.


2) Heat-fix cells by passing the slide rapidly across the flame (do not overheat as it may shrink the
cells and distort the shape)
3) Flood the slide with crystal violet and allow it to react for 1 minute.
4) Tilt the slide and wash with distilled water to remove excess stain (be gentle).
5) Flood the slide with Gram’s iodine and allow reacting for 1 minute.
6) Tilt the slide and wash with distilled water to remove excess stain.
7) Wash the smear with alcohol for 15-20 sec, by holding the slide in vertical position and
observing the release of CV-I complex as blue drops. (This step is done till blue drops stop
appearing).
8) Wash the smear with distilled water.
9) Flood the smear for one minute with safaranin.
10) Tilt the slide and wash with distilled water to remove excess stain (be gentle).
11) Blot off excess water and allow it to dry completely.
12) Carefully position the oil immersion objective over the smear after adding immersion oil to the
smear.

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